Cation conducting GABAA receptors and their use
Granted 16 Oct 2007 · 4 office actions
Current assignee: SANIONA A/S · originally NEUROSEARCH A/S
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Inventors: Philip K. Ahring, Marianne Lerbech Jensen, Thomas Varming · Examiner: Robert C. Hayes · AU 1649 · TC 1600
Life of the patent
3 dated eventsAbstract
This invention relates to cation conducting GABA A receptors, mutated GABA A receptor subunits, polynucleotide sequences encoding mutated subunits, expression vectors comprising the mutated subunits, host cells capable of expressing the mutated subunits, drug screening methods, and chemical substances identified by the drug screening methods of the invention.
Description
10 parts›TECHNICAL FIELD
This invention relates to cation conducting GABA A receptors, mutated GABA A receptor subunits, polynucleotide sequences encoding mutated subunits, expression vectors comprising the mutated subunits, host cells capable of expressing the mutated subunits, drug screening methods, and chemical substances identified by the drug screening methods of the invention.
›BACKGROUND ART
Preferred drug screening method of ion channels include the standard high throughput screens (HTS) using mixtures of test compounds and biological reagents along with indicator compound loaded into cells in arrays of wells, usually in standard microtiter plates with 96 or 384 wells, and measuring the signal from each well, either fluorescence emission, intracellular pH, optical density, radioactivity, etc.
The GABA A receptor is an attractive target for the development of new drugs. The GABA A receptor, however, is a chloride channel, rendering it incompatible with conventional HTS methods. Moreover, most cells capable of functionally expressing GABA A receptors do not maintain a sufficient chloride gradient across their membranes. For validating these targets, cost and time-consuming electrophysiological methods are still the preferred methods.
There is a strong felt need in the art to make these receptors the target for more efficient and inexpensive high throughput drug screening methods.
Wang et al. [Wang Chih-Tien et al: Cation Permeability and Cation-Anion Interactions in a Mutant GABA-Gated Chloride Channel from Drosophila; Biophys. J. 1999 77 691-700] describe an insect GABA receptor that has been made cation permeable by mutation in the near-M2 region. This receptor, however, does not contain modulator sites affected by drugs, and is not suited for high throughput drug screening.
Bertrand et al. [Bertrand D, Galzi J L, Devillers-Thieiy A, Bertrand S & Changeux J P: Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal α7 nicotinic receptor; Proc. Natl. Acad. Sci. USA 1993 90 6971-6975] describe how mutations at two distinct sites within its TM2 domain alter the calcium permeability of a nicotinic α7 receptor.
Keramidas et al. [Keramidas A, Moorhouse A J, French C R, Schofield P R & Barry P H. M2 Pore Mutations Convert the Glycine Receptor Channel from Being Anion- to Cation-Selective; Biophys. J. 2000 78 247-259] describe how three mutations carried out in the M2 transmembrane domain of the chloride-conducting α1 homomeric glycine receptor alter the receptor from being anion- to cation-selective.
A cation-conducting GABA A receptor suited for high throughput drug screening has never been disclosed.
›SUMMARY OF THE INVENTION
It is an object of the present invention to provide a cabon-conducting human GABA A receptor that is suited for high throughput drug screening.
Accordingly, in its first aspect, the invention provides a cation-conducting human GABA A receptor.
In another aspect the invention provides mutated GABA A receptor subunits, comprising one or more mutations in the loop bridging its first and second transmembrane domain (TM1 and TM2).
In yet another aspect the invention relates to polynucleotide sequences encoding the mutated subunit of the invention.
In a fourth aspect the invention provides expression vectors comprising the polynucleotide sequence of the invention.
In a fifth aspect the invention provides host cells comprising the polynucleotide sequence of the invention, or the expression vector of the invention.
In a sixth aspect the invention provides methods of screening chemical compounds for inhibiting, activating or modulating activity of a cation-conducting GABA A receptor, which method comprises the steps of
(i) subjecting a cation-conducting GABA A receptor containing cell to the action of the chemical compound to be screened; (ii) subjecting the cation-conducting GABA A receptor containing cell to activation with GABA or any other GABA-acting substance; and (iii) monitoring ion flux through the cation-conducting GABA A receptor, either directly or indirectly, and thereby determining the action of the chemical compound.
In a final aspect the invention relates to the chemical compounds identified by the method of the invention, and to the use of such compounds for diagnosis, treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is related to GABA A receptor dysfunction.
Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and examples.
›DETAILED DISCLOSURE OF THE INVENTION · 1 of 2
The present invention relates to and provides cation-conducting human GABA A receptors.
The GABA A receptor is the major inhibitory receptor of the central nervous system and is composed of five subunits that assemble to form a chloride channel. To date six main types of GABA A receptor subunits have been described, the α subunit (of which six have been described, i.e. α1-6), the β subunit (of which three have been described, i.e. β1-3), the γ subunit (of which three have been described, i.e. γ1-3), the δ subunit, the ρ subunit (ρ1-3), the ε subunit and the θ subunit. Wild type (wt) GABA A receptors are thought consisting primarily of 2 α-, 2 β- and 1 γ-subunit, but other combinations are also functional, for instance receptors composed only of α and β subunits.
Each GABA A receptor subunit is composed of a large extracellular N-terminal domain, four membrane-spanning domains (TM1-TM4), a small intracellular loop between TM1 and TM2, a small extracellular loop between TM2 and TM3, a large intracellular loop between TM3 and TM4 and a short C-terminal domain. The TM2 domain of each of the five subunits demarcates the chloride-conducting pore of the receptor complex.
According to the present invention it has now been found that a set of mutations introduced in the intracellular loop bridging TM1 and TM2 transmembrane domain, and/or in TM2 itself, affect the physiology of the mutated receptor in a way that the receptor is no longer conducting chloride ions, but rather cations, thereby constituting a cation-conducting GABA A receptor.
In describing the present invention, amino acids are designated using the established one-letter symbols.
Cation-Conducting GABA A Receptors
In its first aspect the invention provides a cation-conducting human GABA A receptor. More specifically the cation-conducting GABA A receptor of the invention may be characterised as being a functional derivative of a wild-type GABA A receptor, derived from the wild-type GABA A receptor by mutation, and thus being a mutated GABA A receptor.
In a preferred embodiment the cation-conducting GABA A receptor of the invention comprises at least one mutated subunit, which subunit holds one or more mutations in the loop bridging its first and its second transmembrane domain (TM1 and TM2), and/or even in the TM2 domain itself.
In a more preferred embodiment the cation-conducting GABA A receptor of the invention comprises at least one mutated α subunit, and/or at least one mutated β subunit, and/or at least one mutated γ subunit. In an even more preferred embodiment the subunit is a mutated α1, α2, α3, α4, α5, α6, β1, β2, β3, γ1 γ2, and/or γ3 subunit.
Mutated GABA A Receptor Subunits
In another aspect the invention provides a mutated GABA A receptor subunit comprising one or more mutations in the loop bridging its first and second transmembrane domain (TM1 and TM2), and/or even in the second transmembrane domain (TM2). The mutated GABA A receptor subunit of the invention may be characterised as being a functional derivative of a wild-type GABA A receptor subunit, derived from the wild-type GABA A receptor subunit by mutation, and thus being a mutated GABA A receptor subunit.
The mutated GABA A receptor subunit of the invention may in particular be a mutated α1, α2, α3, α4, α5, α6, β1, β2, β3, γ1 γ2 or γ3 subunit.
In a preferred embodiment the mutation contemplated according to the present invention should be introduced
at positions corresponding to the position of the partial amino acid sequence represented by “ESVPAR” (SEQ ID NO: 14) in the wild-type GABA A receptor α subunits, at positions corresponding to the position of the partial amino acid sequence represented by “DASAAJR” (SEQ ID NO: 15) in the wild-type GABA A receptor β subunits, or at positions corresponding to the position of the partial amino acid sequence represented by “DAVPAR” (SEQ ID NO: 16) in the wild-type GABAA receptor γ subunits, i.e. at amino acid positions−6′ to−1′ when numbered according to TM2.
In a more preferred embodiment above-identified partial 6 amino acid sequence is substituted for the partial 5 amino acid sequence DSGEK (SEQ ID NO: 17), or any subsequence thereof or any conservative substitution thereof, in the ioop bridging its first and its second transmembrane domain (TM1 and TM2). Expressed another way, the above-identified partial 6 amino acid sequence is substituted for the following partial 5 amino acid sequence:
X 1 X 2 X 3 X 4 X 5 (SEQ ID NO: 18);
wherein
X 1 designates D or E;
X 2
X 3 designates S. C or A;
X 3 designates G, A or V;
X 4 designates E or D; and
X 5 designates K or R.
Most preferred mutants of the invention holds a partial amino acid sequence selected from the following list (SEQ ID NOS: 19-54)
DSGEK, DSGDK, DSAEK, DSADK, DSVEK, DSVDK, DCGEK, DCGDK, DCAEK, DCADK, DCVEK, DCVDK, DAGEK, DAGDK, DAAEK, DAADK, DAVEK, DAVDK, DSGER, DSGDR, DSAER, DSADR, DSVER, DSVDR, DCGER, DCGDR, DCAER, DCADR, DCVER, DCVDR, DAGER, DAGDR, DAAER, DAADR, DAVER, DAVDR.
Polynucleotide Sequences Encoding Mutated Subunits
In another aspect the invention provides a purified and isolated polynucleotide sequence encoding the mutated GABA A receptor subunit of the invention. The mutated GABA A receptor subunit encoded by the polynucleotide sequence of the invention may in particular be a mutated α1, α2, α3, α4, α5, α6, β3, β2, β3, γ1 γ2 or γ3 subunit.
Recombinant Expression Vectors
In a further aspect the invention provides a recombinant expression vector comprising the polynucleotide of the invention.
As defined herein, a recombinant expression vector is an expression vehicle or recombinant expression construct used for introducing polynucleotides into a desired cell. The expression vector may be a virus vector or a plasmid vector, in which the polynucleotide of the invention may be inserted. Suitable expression vehicles include, but are not limited to eukaryotic expression vectors and prokaryotic expression vectors, e.g. bacterial linear or circular plasmids, and viral vectors. However, any other plasmid or vector may be used as long as they are replicable and viable in the production host.
›DETAILED DISCLOSURE OF THE INVENTION · 2 of 2
Preferred eukaryotic expression vectors include pWLNEO, pSV2CAT, pOG44, pXT1, pSG (available from Stratagene); pSVK3, pBPV, pMSG, pSVL (available from Pharmacia); and pcDNA-3 (available from Invitrogen).
The expression vector may further comprise regulatory sequences in operable combination with the polynucleotide sequence of the invention. As defined herein, the term “in operable combination” means that the operable elements, i.e. gene(s) and the regulatory sequences, are operably linked so as to effect the desired expression.
Host Cells
In a yet further aspect the invention provides a host cell comprising the solated polynucleotide sequence of the invention, and/or or a recombinant expression vector of the invention.
The production cell of the invention preferably does not express, or is avoid any endogen GABA A subunit activity.
The production cell of the invention may preferably be a eukaryotic cell, in particular a human cell, or a fungal cell, such as a yeast cell or a filamentous fungal cell. Preferred cells include HEK293, CHO-k1, BHK, COS7, PC12, HiB5, RN33b cell, and a Xenopus laevis oocyte (XLO), or any other cell line able to express the cation-conducting GABA A receptor of the invention.
Methods of Drug Screening
The cation-conducting GABA A receptor of the invention allows for the first time the drug screening process to be carried out using conventional high-throughput screening technologies.
Therefore, in another aspect the invention provides a method for the screening of chemical compounds for GABA A receptor activity, by which method a chemical compound having GABA A receptor activity is identified by its ability to inhibit, activate or modulate the flux of ions through the GABA A receptor, to change the intracellular pH, or to change the membrane potential of a cation-conducting GABA A receptor containing cell.
In a preferred embodiment the method of the invention comprises the steps of
(i) subjecting a cation-conducting GABA A receptor containing cell to the action of the chemical compound to be screened; (ii) subjecting the cation-conducting GABA A receptor containing cell to activation with GABA or any other GABA-acting substance; and (iii) monitoring ion flux through the cation-conducting GABA A receptor, either directly or indirectly, thereby determining the action of the chemical compound.
The Cation-Conducting GABA A Receptor Containing Cell
The cation-conducting GABA A receptor containing cell used in the method of the invention preferably is a host cell of the invention as described above.
Monitoring of the Ion Flux
According to the method of the invention, the ion flux through the cation-conducting GABA A receptor is monitored in order to determine inhibition, activation or modulation of ion flux caused by the chemical compound. The ion flux may be monitored directly or indirectly using established methods.
In a preferred embodiment monitoring of the flux through the cation-conducting GABA A receptor is performed using fluorescence or radio-ligand methods.
In a more preferred embodiment the cabon-conducting GABA A receptor containing cell is loaded or incubated with a fluorescence dye or radio-ligand that allows for a determination of changes in ion flux through the cation-conducting GABA A receptor caused by the addition of the chemical test substance and GABA or the GABA-acting compound.
Preferred fluorescence indicators include, but are not limited to FLUO-3, FLUO4, Calcium Green, FURA-2, SBFl, PBFl, CD222, and BCECF, DIBAC 4 (3), DiOC5(3), and DiOC2(3).
Preferred radioligands include, but are not limited to Rb + and organic cations such as TPP + .
In yet another preferred embodiment monitoring of ion flux through the cation-conducting GABA A receptor may be performed by spectroscopic methods, e.g. using a FLIPR assay (Fluorescence Image Plate Reader; available from Molecular Devices), or by using the automated analysis equipment described in U.S. Pat. No. 5,670,113.
In yet another preferred embodiment monitoring of the ion flux through the cation-conducting GABA A receptor is performed by patch clamp techniques, e.g. as described by Hamill, O. P., et al., Pflüigers Arch. 1981 351 85-100. In a more preferred embodiment, monitoring of the membrane potential of the cation-conducting GABA A receptor containing cell is performed by the automatic patch clamp method described in WO 98/50791.
Cation-Conducting GABA A Receptor Active Compounds
In another aspect the invention relates to chemical compounds identified by the method of the invention, and capable of inhibiting, activating or modulating GABA A receptors.
The chemical compounds of the invention are useful for the diagnosis, treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is related to GABA A receptor dysfunction. In a preferred embodiment, the disease, disorder or condition is asthma, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, allergies, benign prostatic hypertrophy, prostate cancer, Parkinson's disease, psychotic and neurological disorders, anxiety, schizophrenia, mania, depression, dyskinesia, memory disorders, sleep disorders, convulsive disorders, and epilepsy.
›EXAMPLES
The invention is further illustrated with reference to the following examples, which are not intended to be in any way limiting to the scope of the invention as claimed.
›Examples4
›Example 1
Mutagenesis and Expression of Mutated Receptors
To investigate the influence of the GABA A receptor subunits in defining the ion selectivity of the receptor, a mutant GABA A receptor subunit β 3 was designed by site directed mutagenesis.
The GABA A receptor subunits were cloned by RT-PCR from human brain mRNA (Clontech). The GABA A receptor α subunit was sub-cloned into the pNS1z vector, the GABA A receptor β subunit was sub-cloned into the pNS1n vector, and the GABA A receptor γ subunit was sub-cloned into the pZeoSV vector (Invitrogen). pNS1n and pNS1z were derived from pcDNA3-Neo (Invitrogen) or pcDNA3-Zeo (Invitrogen), respectively.
A hindIII site was introduced in the loop between the M1 and M2 domains and a bsu36I site was introduced in the loop between the M2 and M3 domains in all receptor subunits. hindIII and bsu36I sites at other positions within the vectors or cDNAs were eliminated.
A set of chimeric GABA A R subunits was constructed in which the M2 domain was moved from one subunit to another by simple restriction digestion and ligation. The GABA A R encoding plasmids were digested by the restriction enzymes hindIII and bsu36I, and fragments were then separated using gel-electrophoresis and purified using a gel extraction kit (Qiagen). Chimeric GABA A R subunits were obtained by ligation of the fragments using the rapid ligation kit (Roche).
To create chimeric GABA A Rs with non-GABA M2 domains, overlapping oligonucleotides encoding the entire M2 domains were annealed and elongated using Expand DNA polymerase.
The elongation conditions were as follows: 94° C. for 2 minutes, followed by 20 cycles of 94° C. for 1 minute, 59° C. for 1 minute and 72° C. for 1 minute, and ending with an incubation at 72° C. for 10 minutes. The synthetic M2 domains were digested with hindIII and bsiB6I and ligated with the cDNA of a GABA A R subunit.
Mutations were introduced into the cDNA's of the GABA A receptor β 3 subunit using the QUANT-ESSENTIAL SDM kit (Quantum Biotechnologies). A block of 6 amino acids (DASAAR) (SEQ ID NO: 15) at the TM2 border of the β 3 subunit was replaced by the corresponding amino acids of the AChRα 7 (DSGEK) (SEQ ID NO: 19).
The mutagenesis was performed using the following oligonucleotide primers:
To create further mutated GABA A R β 3 subunits the following oligonucleotide primers were used:
Introduction of the bsu36I site in the hGABA A R β 3 subunit led to the I300V mutation, which turned out to affect receptor function. This was reversed using the following oligonucleotide primer:
Correct mutagenesis was verified by restriction enzyme analysis and by DNA sequencing. CHO-k1 cells (ATCC) were co-transfected with the plasmids described above and a plasmid encoding enhanced green fluorescent protein (GFP, Clontech), using the lipofectamine PLUS kit (Life Technologies).
›Example 2
Expression of Mutated Receptors
All constructs were expressed in CHO-K1 cells (ATCC No. CCL61).
CHO-KL cells were maintained in DMEM with 10 mM HEPES and 2 mM glutamax supplemented with 10% fetal bovine serum and 2 mM L-proline (Life Technologies). The cells were cultured at 37° C. in a humidified atmosphere of 5% CO 2 and 95% air and passaged twice a week.
CHO-K1 cells were co-transfected with the plasmids described above and a plasmid encoding enhanced green fluorescent protein using the lipofectamine PLUS kit (Life Technologies) according to manufacturers protocol.
Binding experiments and electrophysiological measurements were performed 24-48 hours after transfection.
›Example 3
Binding Assays
Binding studies were performed using standard methods. Membranes were prepared from CHO-K1 cells expressing recombinant GABA A R subunits. The cells were washed in PBS (Life Technologies), trypsinized, washed twice in tris-citrate buffer (50 mM, pH 7.1) and centrifuged for 10 minutes at 5,000 g.
[ 3 H]-Muscimol Binding
Membranes were resuspended in membrane wash buffer [20 mM KH 2 PO 4 K 2 HPO 4 , pH 7.5, 50 mM KCl, 0.025% (w/v) NaN 3 , and various protease inhibitors (1 mM EDTA, 2 mM benzamidine chloride, 0.1 mM benzethonium chloride, 50 U/ml bacitracin, 0.3 mM phenylmethylsulfonyl fluoride, 10 mg/I ovomucoid trypsin inhibitor, 10 mg/l soybean trypsin inhibitor)] and centrifuged for 30 minutes at 177,000 g and 4° C. The pellet was resuspended in binding assay buffer (20 mM KH 2 PO 4 /K 2 HPO 4 , pH 7.5 and 100 mM KCl) to a protein concentration of 1 mg/ml and homogenized just before use. Binding was performed with 1, 3, 10, 30, 100 or 300 nM of [ 3 H]-muscimol (20 Ci/mmol, Dupont-New England Nuclear) in triplicate in a final volume of 250 μl containing 200 μg of protein, and non-specific binding was determined in the presence of 1 mM GABA (Sigma). Samples were incubated at 4° C. for 30 minutes and labelled membranes were harvested on a Brandel cell harvester using GF/B filters (Whatman). The filters were washed with 3×4 ml binding assay buffer and the amount of radioactivity was determined by liquid scintillation counting.
[ 3 H]-Flumazenil Binding
Membranes were resuspended in tris-citrate buffer and centrifuged for 10 minutes at 22,000 g at 4° C. The pellet was resuspended in tris-citrate buffer to a protein concentration of 100-200 μg/ml. Binding was performed with 0.5, 1, 2, 3 or 6 nM [ 3 H]-flumazenil (87 Ci/mmol, Dupont-New England Nuclear) in triplicate in a final volume of 550 μl containing 50-100 μg protein, and non-specific binding was determined in the presence of 1 μM clonazepam (Roche). Samples were incubated at 4° C. for 40 minutes and labelled membranes were harvested using rapid filtration over GF/C filters (Whatman). The filters were washed with 2×5 ml tris-citrate buffer and the amount of radioactivity was determined by liquid scintillation counting.
[ 35 S]-TBPS Binding
Membranes were resuspended in tris-citrate buffer and centrifuged for 10 minutes at 22,000 g at 4° C. The pellet was resuspended in 20 mM KH 2 PO 4 buffer (pH 7.4) containing 200 mM KCl to a protein concentration of 200-400 μg/ml. Binding was performed in triplicate with 5 nM [ 35 S]-TBPS (89 Ci/mmol, Dupont-New England Nuclear) and 0.6 μM GABA in a final volume of 550 μl containing 100-200 μg of protein, and non-specific binding was determined in the presence of 200 μM picrotoxin (Sigma). Samples were incubated at 20-220° C. for 150 minutes and binding was terminated by rapid filtration over Whatman GF/C filters. The filters were washed with 2×5 ml 20 mM KH 2 PO 4 buffer (pH 7.4) containing 200 mM KCl and the amount of radioactivity was determined by liquid scintillation counting.
(+) indicates more and (÷) indicates less than 2.5% binding of the wt receptor.
Data Analysis
K d and B max were estimated using a one site binding equation.
The results of this experiment are presented in Table 1, below.
›Example 4
Characterisation
The mutant GABA A receptor subunit β3 obtained according to Examples 1-2 was characterized electrophysiologically with respect to reversal potentials.
All experiments were performed in voltage clamp using conventional whole cell patch clamp methods. The amplifier was an EPC-9 (HEKA-electronics, Lambrect, Germany) run by a Macintosh G3 computer via an ITC-16 interface. Experimental conditions were set with the Pulse-software accompanying the amplifier. Data were low pass filtered and sampled directly to a hard disk at a rate of 3 times the cut-off frequency.
Pipettes were pulled from borosilicate glass (Modulohm) using a horizontal electrode puller (Zeitz-Instrumente). The pipette resistances were 1.6-2.6 mΩ and the pipettes were filled with a solution containing 120 mM KCl 2 mM MgCl 2 , 10 mM EGTA and 10 mM HEPES adjusted to pH=7.2. The pipette electrode was a chloridized silver wire, and the reference was a silverchloride pellet electrode (In Vivo Metric) fixed to the experimental chamber. The electrodes were zeroed with the open pipette in the bath just prior to sealing. Coverslips with cultured cells were transferred to a 15 μl experimental chamber mounted on the stage of an inverted microscope (IMT-2, Olympus) supplied with Nomarski optics and a mercury lamp (Olympus). Cells chosen for experiments emitted bright green fluorescence when exposed to UV-light from the mercury lamp. After giga-seal formation the whole cell configuration was attained by suction.
Cells were continuously superfused at a rate of 2.5 ml/min with an extracellular solution containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 10 mM HEPES adjusted to pH=7.4. A low chloride extracellular solution (Gluconate-R) containing 5 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 135 mM NaGluconate and 10 mM HEPES adjusted to pH=7.4 was used to address the chloride permeability and a low sodium extracellular solution (NMDG-R) containing 14 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 126 mM NMDG and 10 mM HEPES adjusted to pH=7.4 with HCl was used to address the cation permeability.
The cells were held at a holding potential of−60 mV at the start of each experiment and the current was continuously measured for 30 sec to ensure a stable baseline. The I-V experiments were performed by holding the cells at potentials of −60, −50, −40, −30, −20, −10, 0, 10, 20, 30, 40, 50, 60 mV and recording the GABA-activated currents at each membrane potential.
GABA-containing solutions were delivered to the chamber through a custom-made gravity-driven flowpipe, the tip of which was placed approximately 50 μm from the cell. Application was triggered by compression of the tubing connected to the flow pipe with a valve controlled by the Pulse-software. In general, GABA was applied for 0.5-1 sec every 30-40 sec. The sampling interval during application was 1 msec. The patch clamp experiments were performed at room temperature (20-22° C.). Currents were measured at the peak of the response and reversal potentials (V rev ) were read directly from the I-V plots for each cell.
Wild-type and the mutated GABA A receptors were characterized concerning reversal potentials in different extracellular solution to identify cation—or anion conductance.
The results of this experiment are presented in Table 1, below.
As shown in Table 1, the shift in reversal potential from Na—R to Gluconate-R indicates chloride conductance for the wild-type receptor, but not for the mutant receptor, whereas the shift in reversal potential from Na—R to NMDG-R indicate cation conductance for the mutant receptor, but not for the wild-type receptor.
No changes are observed for [ 3 H]muscimol- and [ 3 H]flumazenil binding, indicating that the receptor still is a functional GABA A receptor, whereas binding of the GABA A channel blocker [ 35 S]TBPS is abolished by the mutation, indicating a modification of the channel.
›Tables in the description — 3
| 5′ GACTCGGGCG AGAGAGTTGC CCTCGGGATC 3′ | (SEQ ID NO: 10) |
| hGABA β 3 G-EK s | |
| 5′ CTGGATCAAT TATGACGCAG GCGAGAAGGT TGCC 3′ | (SEQ ID NO: 11) |
| hGABA β 3 G-E s | |
| 5′ CTGGATCAAT TATGACGCAG GCGAGAGAGT TGCCCTCGGG ATC 3′ | (SEQ ID NO: 12) |
| 5′ GAGACCTTGC CTAAGATCCC | (SEQ ID NO: 13) |
| CTATGTCAAA GCC 3′ |
| V rev | Kd | [ 3 H] | ||||||
| V rev | V rev | Gluconate- | [ 3 H] | Flu- | [ 35 S] | |||
| α 2 | Na-R | NMDG-R | R | Muscimol | mazenil | TBPS | ||
| sub- | β 3 | γ 2 | [mV ± | [mV ± | [mV ± | binding | binding | bind- |
| unit | subunit | sub-unit | SEM] | SEM] | SEM] | nM | nM | ing* |
| α 2 | β 3 | γ 2 | 1.3 ± 0.7 | 0.8 ± 2.0 | 33.5 ± 2.4 | 54.7 ± 14.7 | 2.4 ± 0.7 | + |
| α 2 | β 3 | γ 2 | −2.7 ± 0.6 | −31.4 ± 1.9 | −9.5 ± 0.7 | 38.0 ± 6.0 | 1.8 ± 0.6 | ÷ |
| DSGEK | ||||||||
| α 2 | β 3 | γ 2 | −3.8 ± 1.5 | −35.1 ± 4.3 | ND | ND | ND | ND |
| DSGEK | DSGEK | |||||||
| *(+) indicates more and (÷) indicates less than 2.5% binding of the wt receptor | ||||||||
| ND = not determined |
Claims
14 · 3 independent · depth 3Classifications
34 codes- A61P25/28
- A61P9/04
- A61P25/00
- A61P11/10
- A61P9/10
- A61P25/22
- A61P1/04
- A61P25/24
- A61P43/00
- A61P25/20
- A61P37/08
- A61P25/16
- A61P35/00
- A61P25/08
- A61P13/08
- A61P9/02
- A61K38/17
- A61P11/06
- A61P9/12
- A61K45/00
- A61P25/18
- A61P13/02
- C12N15/09
- C12N1/21
- C07K14/705
- C12N5/10
- C12N1/15
- C07K14/00
- C12N1/19
- C12Q1/02
- G01N33/60
- G01N33/53
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1 priority documents›Priority documents — 1
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| related publication | US 20050049190 A1 | 3 Mar 2005 |
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11 members · 7 offices›IP5 & PCT — 8 members
| Office | Publication | Kind | Published | Filed | Status | Title |
|---|---|---|---|---|---|---|
| US | US-2005049190-A1 | A1 | 3 Mar 2005 | 4 Jun 2002 | published | Cation conducting gabaa receptors and their use |
| USthis patent | US-7282344-B2 | B2 | 16 Oct 2007 | 4 Jun 2002 | granted | Cation conducting GABAA receptors and their use |
| US | US-2008033061-A1 | A1 | 7 Feb 2008 | 14 Aug 2007 | published | Cation conducting gabaa receptors and their use |
| EP | EP-1399479-A2 | A2 | 24 Mar 2004 | 4 Jun 2002 | published | Kationenleitende gaba-a-rezeptoren und deren verwendungde |
| EP | EP-1399479-B1 | B1 | 16 Sep 2009 | 4 Jun 2002 | granted | Kationenleitende gaba-a-rezeptoren und deren verwendungde |
| JP | JP-2005506056-A | A | 3 Mar 2005 | 4 Jun 2002 | published | カチオン伝導ｇａｂａａレセプターおよびその使用ja |
| WO | WO-02098907-A2 | A2 | 12 Dec 2002 | 4 Jun 2002 | published | Cation conducting gabaa receptors and their use |
| WO | WO-02098907-A3 | A3 | 24 Apr 2003 | 4 Jun 2002 | published | Recepteurs gabaa conduisant les cations et utilisationfr |
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| Office | Publication | Kind | Published | Filed | Status | Title |
|---|---|---|---|---|---|---|
| AT | AT-E443083-T1 | T1 | 15 Oct 2009 | 4 Jun 2002 | granted | Kationenleitende gaba-a-rezeptoren und deren verwendungde |
| AU | AU-2002304915-A1 | A1 | 16 Dec 2002 | 4 Jun 2002 | published | Cation conducting gabaa receptors and their use |
| DE | DE-60233728-D1 | D1 | 29 Oct 2009 | 4 Jun 2002 | granted | Kationenleitende gaba-a-rezeptoren und deren verwendungde |
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